Protein Protocols Protein Protocols

Zn 2+ -Reverse Staining 253
Fig. 1. Reverse staining of rat brain homogenate proteins (70 µg load) after 2-D-PAGE.
Proteins in a wide range of molecular weights and isoelectric points are detected as transparent
spots. These spots contain protein-SDS complexes that bind Zn 2+ and thereby inhibit the precipitation
of ZnIm 2 locally.
45 s or band overstaining and loss of the image will occur. Overstaining is prevented by
pouring off the developer solution and rinsing the gel 3–5× (~1 min) in excess water
(see Note 3).
At this point, the reverse stained gel can be photographed (Fig. 1). Photographic
recording is best conducted with the gel placed on a glass plate held a few centimeters
above a black underground and under lateral illumination.
While SDS-PAGE is a popular, high-resolution method for separating complex protein
mixtures, sometimes it is desired to avoid either protein denaturation or disruption
of macromolecular complexes during electrophoresis. In this case, the proteins are separated
in the absence of SDS (native PAGE or agarose gel electrophoresis). Therefore, it
is desirable to avoid the use of SDS during the reverse staining step. Two procedures
have been developed to resolve this problem (see Notes 8 and 9; Figs. 2 and 3).
4. Notes
1. Imidazole is a five-membered heterocyclic ring containing a tertiary (“pyridine”) nitrogen
at position 3, and a secondary (“pyrrole”) nitrogen at position 1. It is a monoacidic base
whose basic nature is due to the ability of pyridine nitrogen to accept a proton. The pyrrole
nitrogen can lose the hydrogen atom producing imidazolate anion (Im – ), at high pH values
(pKa ~ 14.2). However, deprotonation of imidazole’s pyrrolic nitrogen may also occur at
lower pH values, upon complexation of Zn2+ at the pyridinium nitrogen (ref. 9 and references
therein). As a result, ZnIm2 can form and precipitate at pH > 6.2. Upon treatment of